The present invention relates to highly efficient and highly repressible expression control sequences, expression vectors which contain these expression control sequences, microorganisms transformed with these expression vectors, and methods for their production by means of recombinant DNA technology. The present invention also relates to methods for the production of viral, prokaryotic, and eukaryotic proteins and fragments thereof using these highly repressible expression control sequences, expression vectors and transformed microorganisms.
The level of production of a protein in a host cell is determined by three major factors: the number of copies of its structural gene within the cell, the efficiency with which the structural gene copies are transcribed and the efficiency with which the resulting messenger RNA (xe2x80x9cmRNAxe2x80x9d) is translated. The transcription and translation efficiencies are, in turn, dependent on nucleotide sequences which are normally situated ahead of the desired structural genes or the translated sequence. These nucleotide sequences (expression control sequences) define, inter alia, the location at which the RNA polymerase binds (the promoter sequence to initiate transcription; see also EMBO J. 5:2995-3000 (1986)) and at which the ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation.
Not all expression control sequences have the same efficiency. It is therefore often advantageous to separate the specific coding sequence for a desired protein from its adjacent nucleotide sequences and to link it with other expression control sequences to achieve a higher expression rate. After this linkage has been accomplished, the newly combined DNA fragment can be inserted into a plasmid capable of achieving a high copy number or a derivative of a bacteriophage to increase the structural gene copies within the cell, thereby improving the yield of the desired protein.
The constitutive expression of overproduction of both toxic and normally nontoxic gene products is often harmful to the host cells, thereby, lowering the stability of a specific host cell-vector system. Therefore, an expression control sequence should, in addition to improving the transcription and translation efficiency of a cloned gene, be regulatable to permit the regulation of the expression during the growth of the microorganisms. Some regulatable expression control sequences can be switched off during the growth of the host cells and then can be switched on again at a desired point in time, to favor the expression of large amounts of the desired protein.
Various expression control sequences have been used to regulate the expression of DNA sequences and genes which code for desired proteins. See, e.g., Itakura et al.(1977) Science 198:1056-1063; Goeddel et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:106-110; Emtage et al. (1980) Nature 283:171-174; Bernard et al. (1979) Sciences 205:602-607; Ammann et al. (1983) Gene 25:167-178; de Boer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25; European Patent Application Publication Nos. 41767 and 186069.
In accordance with the invention, highly efficient and highly repressible expression control sequences can be produced by combining promoter sequences with operator/repressor sequences. The present invention provides for expression control sequences for the expression of polypeptides from a gene or polynucleotide of interest. The gene or polynucleotide may express a viral, prokaryotic, or eukaryotic polypeptide. These control sequences are produced by combining bacterial or phage promoter and operator/repressor systems.
More particularly, the present invention provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a xe2x88x9230 region, having a sequence represented by the sequence 5xe2x80x2 TTGMYW 3xe2x80x2 (where M=A or C and Y=C or T and W=A or T), and a xe2x88x9212 region, having a sequence represented by the sequence 5xe2x80x2 TAWRMT (where R=A or G).
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a xe2x88x9230 region, having a sequence represented by the sequence 5xe2x80x2 TTGAYA 3xe2x80x2 (where Y=C or T), and a xe2x88x9212 region, having a sequence represented by a sequence selected from the group consisting of: 5xe2x80x2 TAWRTT 3xe2x80x2 (where R=A or G), 5xe2x80x2 TAWGMT 3xe2x80x2 (where W=A or T and M=A or C), and 5xe2x80x2 TAARMT 3xe2x80x2.
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a xe2x88x9230 region, having a sequence represented by the sequence 5xe2x80x2 TTGMTW 3xe2x80x2 (where M=A or C and W=A or T), and a xe2x88x9212 region, having a sequence represented by a sequence selected from the group consisting of: 5xe2x80x2 TAWRTT 3xe2x80x2 (where R=A or G), 5xe2x80x2 TAWGMT 3xe2x80x2, and 5xe2x80x2 TAARMT 3xe2x80x2.
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a xe2x88x9230 region, having a sequence represented by the sequence 5xe2x80x2 TTGCTW 3xe2x80x2 (where Y=C or T and W=A or T), and a xe2x88x9212 region, having a sequence represented by a consensus sequence selected from the group consisting of: 5xe2x80x2 TAWRTT 3xe2x80x2 (where R=A or G), 5xe2x80x2 TAWGMT 3xe2x80x2 (where M=A or C), and 5xe2x80x2TAARMT 3xe2x80x2.
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a xe2x88x9230 region, having the sequence 5xe2x80x2 TTGACT 3xe2x80x2, and a xe2x88x9212 region, having the sequence 5xe2x80x2 TAAGAT 3xe2x80x2.
The present invention further provides for expression vectors comprising the above expression control sequences, host cells transformed with such expression vectors, and methods for producing viral, prokaryotic, and eukaryotic polypeptides using the disclosed expression control sequences.